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Issn: 2277–4998 IJBPAS, November, 2015, 4(11), Special Issue: 01-11 ISSN: 2277–4998 INVESTIGATING THE KARYOTYPE OF FIVE POPULATIONS OF “PLANTAGO MAJOR”SPECIES USING THE IMAGE ANALYSIS SYSTEM FATEMEH AGHAJANI1, ARMAN MAHMOUDI OTAGHVARI2*, SADEGH POURMORADI3, SEDIGHEH KELIJ4 1: M.Sc. Student, Department of Biology, University of Mazandaran, Babolsar, Iran 2: Department of Biology, University of Mazandaran, Babolsar, Iran 3: Mazandaran Agricultural Research and Natural Resources Center, Mazandaran, Iran 4: Department of Biology, University of Mazandaran, Babolsar, Iran *Corresponding Author: E Mail: [email protected] ABSTRACT With the aim of investigating the Cytotaxonomy of a species of Plantago genus from the family of Plantaginaceae, collected from the habitats of the northern provinces of Iran, the karyotype of 5 populations belonging to P.major species is studied in the meristem cells of root tip using an image analysis system. The chromosomal dimensions such as the length of short arm and the length of the whole chromosome are measured and the AR and CI statistics are also calculated after preparation, staining, and microscopic investigation of samples and separation of chromosomes by Photoshop software through Micromeasure software in at least 3 appropriate cells of mitotic metaphase. The populations are measured based on the karyotype statistics including S%, %TF, A1, DRL, A2, VRC, Levan karyotype formula and grouping the Stebbins for studied populations. In this research, the studied populations of P.major had the chromosomal number of 2n=2x= 12 and the basic chromosome number was equal to x=6 in all populations and it was consistent with previous studies and this indicates the diploid species in Iran. Furthermore, according to the Levan Table, the chromosomes of studied populations were from the metacentric and submetacentric species. Keywords: Plantago major , Cytogenetic Diversity, Chromosome, Karyotype, Northern Iran INTRODUCTION Plantago genus has about 483 species [21], perennial species for it. It has the dense and 22 species of this genus have been reported alternate Rosulates. The male and female from Iran in Flora Iranica (Mozaffarian, flowers are inside the spikes until a narrow 1994). Most of its plants are have one or and extremely long cylinder with a few or a multiple years of age and there are a few lot of flowers [11],The application of 1 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article cytogenetic information in systematic and population of a species because different phylogenetic nature of this genus has been populations of a species show their unique considered from previous years. Rana genomic consistency with the environment (1996) has offered a new suggestion of wherein they grow, so that it is believed classifying the Plantago genus based on the that the chromosomal analyses along with cytology knowledge and classified this the genetic and morphological studies can genus into 6 subgenus including Coronopus be the reliable indices in assessing the Rahn, Albicans Rahn, Plantago major, relative relationship between the species of Psyllium Juss, Littorella Rahn, and a genus [8],The investigation of difference Bougueria Rahn, and has indicated that in the number of chromosome, its Plantago genus has 3 different chromosome morphology as well as the difference of bases including x= 4, x= 5, and x = 6. ploidy level are utilized as the evidence for The cultivation of this genus has been more convergence between the species and taken into account due to the important somatic hybrids derived from the cell components (they are more applied in fusion [13],A lot of international studies are pharmacy) in order to extract the active conducted by different researchers on compounds [7],Plantago major has the determining the chromosome number and biological active compounds like ploidy level of species and subspecies of a Polysaccharide, Glycoside, Lipid and Plantago genus. McCullagh [14],has Terpenoid, and Sorbitol [18], It has the presented the first chromosome counting astringent, anti-diarrhea, sweat-inducing, and morphology of chromosomes. Gorenf, diuretic, and anti-rheumatism effects Vasudevan and Favarger (1972) studied the (Zargari, 1992). It is effective in curing the polyploidy in this species. Santosh and colic, jaundice, and liver diseases (Beara, Raghbir (2011) investigated the 2000). Furthermore, it has the anti- chromosome number of some species of inflammatory on the liver. (Turel et al, Plantago genus in the northern India. 2009) and it is especially applied in wound Conducting the Cytotaxonomic study of healing[2], Plantago major is spread in this genus in the northern Iran, we will be different parts of the Earth and grows in a able to provide the more accurate and large area of Europe, Asia, and North detailed classification of this Taxon and America [22],and [6],It is growing in provide the better and more effective key of almost all regions of Iran. It is essential to identification by discovering the conduct the karyotype studies in the chromosomal status of plantago major 2 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article species populations in the north of country 16-18 hours for stabilization of internal in addition to determining the level of structure of cell and maintaining the karyotype symmetry. original status as well as HCl 1N for MATERIALS AND METHODS hydrolysis for 5-10 minutes at 60°C and This study is conducted on five accessions also ethyl alcohol 70% for storing the roots from P. major species in different regions for a long time in addition to hematoxylin of northern Iran (Gorgan, Behshahr, Sari, staining solution in order to investigate the Babolsar, and Babol). The studied seeds shape and structure of chromosomes. germinate after disinfection by bleach 15% Afterwards, the apical meristem of roots is for ten minutes and putting them on the wet separated and the chromosomal images are filter paper inside the Petri dishes at 25 °C, taken by adding acetic acid 45% and and the root tips are separated from the through the squash method (Agayev, 1996). seeds when the root lengths reach 0.5-1 cm The chromosomes of each cell are and are put as the pretreatment in solution separated by Photoshop software and of Alpha Bromonaphthalene 1% for 2 arranged on a page, and then the length of hours. The pretreatment refers to stopping long arm, length of short arm, and total the cell division at the metaphase stage. length of chromosome are measured in at The next stages of this study apply the least 3 appropriate mitotic metaphase cells compounds such as the Farmer solution for through Micromeasure software. Table 1: Characteristics of species and populations applied in this research Species Collection place Height above sea level P.major Mazandaran - Sari 1900 P.major Mazandaran - Babol 2577 P.major Mazandaran - Behshahr 400-660 P.major Gorgan 3500 P.major Mazandaran - Babolsar -21 The data is analyzed in the form of a characteristics including the length of long completely randomized unbalanced and short arms are calculated for different method, and then the mean of each populations through Excel software and the characteristic is compared by Duncan test ideogram of each population drawn. The at the level of 5%. The karyotype statistics chromosomes are arranged based on the S%, %TF (Huziwara, 1962), A1, DRL, A2 reduced length of chromosome and the (Romrozarco, 1971), Levan karyotype centromeres aligned in drawing the formula (1962) and Symmetry Class ideogram. (Stebbins, 1971) are measured for studied RESULTS populations and the populations compared The mitotic metaphase images of studied with each other. The mean chromosomal species and populations are presented in 3 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article Figure 1. All of the chromosomes are not and short arms are calculated through Excel clearly seen in an image since they are software and the ideogram of each one multilevel. The mean chromosomal drawn. (Figure 1) characteristics including the length of long Babol P.major Sari P.major Gorgan P.major 4 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article Behshahr P.major Babolsar P.major Figure 1: The mitosis metaphase image and karyogram of studied populations of Plantago major species In drawing the ideogram of species, the difference between the differences in the chromosomes are arranged based on relative length (DRL) of chromosomes, reducing the total chromosome length and Pearson dispersion coefficient (A2) as the the centromeres placed in the same inter-chromosomal asymmetry index, direction. The parameters including the Value of Relative Chromatin (VRC), Levan relative length of shortest chromosome karyotype formula and Stebbins grouping (S%), the total form percentage (%TF), A1 are measured for species and presented in (intra-chromosomal asymmetry index), the Table 2. Table 2: Measurement parameters of karyotype symmetry for five studied populations from Plantago major species Karyotype Population %S %TF A1 DRL A2 VCR SC formula Babol 14.21 38.40 0.36 4.78 0.10 4.05 1A 5m + 1sm Sari 13.54 39.51 0.348 6.87 0.14 3.51 1A 6m Behshahr 14.46 38.87 0.35 4.62 0.21 3.78 1A 5m + 1sm Babolsar 12.63 38.31 0.38 7.56 0.20 3.32 1A 6m Gorgan 13.27 36.46 0.42 5.94 0.13 4.25 1A 5m + 1sm The results indicate that Babol proportion chromosomal base is x=6. The lengths of has 2n= x2x=12 chromosomes and its its longest and shortest chromosome are 5 IJBPAS, November, 2015, 4(11), Special Issue Fatemeh Aghajani et al Research Article equal to 4.61 and 3.46 respectively. In this arms 14.97 μm, and the total length of short species, the maximum and minimum arms 9.33 μm. This species with the lengths of long arm (L) are equal to 2.04 karyotype formula of 5m+1sm is in class and 2.88, the lengths of short arm (S) equal A1 in terms of karyotype symmetry and to 1.40 and 1.73, the ratio of lengths of long based on Stebbins table, and the average to short arm of chromosome (AR) equal to length of each chromosome is 4.05 μm.
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